Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention provides methods and compositions for inhibiting
p53-inactivated cancers. Cancer cells are preferentially inhibited
compared to normal cells by inhibiting tumor survival kinases that are
required for growth of tumor cells but not normal cells.

Claims:

1. (canceled)

2. The method of claim 5, wherein said inhibitor comprises the general
structure of PAK3 inhibitor Chemotype 4, and wherein said inhibitor is
selected from the group consisting of LDN-0211958, LDN-0211959,
LDN-0026056, LDN-0211955, LDN-0041012, and LDN-0028618.

3. (canceled)

4. The method of claim 5, wherein said inhibitor comprises the general
structure of PAK3 inhibitor Chemotype 8, and wherein said inhibitor is
LDN-0044878 or LDN-0091420.

6. A method of inhibiting proliferation of or killing a p53-deficient
cell, comprising contacting said cell with a composition comprising an
inhibitor of PAK3, wherein said inhibitor comprises LDN-0047862,
LDN-0009460, LDN-0042112, LDN-0097519, LDN-0096422, LDN-0111371,
LDN-0086947, LDN-001731, LDN-0080086, or LDN-0097728.

7. (canceled)

8. The method of claim 13, wherein said inhibitor comprises the general
structure of SGK2 inhibitor Chemotype 1A, and wherein said compound is
LDN-0149188.

9. (canceled)

10. The method of claim 13, wherein said inhibitor comprises the general
structure of SGK2 inhibitor Chemotype 2A, and wherein said inhibitor is
selected from the group consisting of LDN-0144705 and LDN-0144676.

13. A method of inhibiting proliferation of or killing a p53-deficient
cell, comprising contacting said cell with a composition comprising an
inhibitor of SGK2, wherein said inhibitor comprises a general structure
selected from SGK2 inhibitor Chemotypes 1, 1A, 1B, 2, 2A, and 4.

14. A method of inhibiting proliferation of or killing a p53-deficient
cell, comprising contacting said cell with a composition comprising an
inhibitor of SGK2, wherein said inhibitor comprises LDN-0181476 or
LDN-0187289.

15. The method of claim 5, 6, 13 or 14, wherein said cell is a p53
deficient tumor cell.

16. The method of claim 5, 6, 13 or 14, wherein said cell is a human
papilloma virus (HPV)-infected cell.

17. The method of claim 5, 6, 13 or 14, wherein said cell is a non-tumor
cell expressing an HPV oncoprotein.

18. The method of claim 5, 6, 13 or 14, wherein said cell is a tumor cell
or tumor cell line of a tissue type selected from the group consisting of
breast, cervix, uterus, bladder, brain, lung, esophagus, liver, and
prostate.

19. A method of identifying an anti-tumor agent for inhibition of p53
deficient tumor cells, comprising contacting tumor survival kinase with a
candidate compound and determining whether said candidate compound
inhibits enzymatic activity of said kinase, wherein a reduction in a
level of said activity in the presence of said candidate compound
compared to that in the absence of said candidate compound indicates that
said candidate compound preferentially inhibits p53 deficient tumor
cells.

20. A method of identifying an anti-tumor agent for inhibition of p53
deficient tumor cells, comprising contacting a cell dependent upon a
tumor survival kinase with a candidate compound and determining whether
said candidate compound inhibits survival or proliferation of said cell,
wherein a reduction in a level of said survival or proliferation in the
presence of said candidate compound compared to that in the absence of
said candidate compound indicates that said candidate compound
preferentially inhibits p53 deficient tumor cells.

21. The method of claim 19 or 20, wherein said tumor survival kinase is
selected from the group consisting of a serum- and glucocorticoid-induced
protein kinase (SGK), a p21-activated kinase (PAK), or a cyclin-dependent
protein kinase (CDK).

22. The method of claim 21, wherein said SGK is SGK2, wherein said PAK is
PAK3, and wherein said CDK is CDK7.

23. A method of identifying a tumor survival kinase, comprising
synthetically inhibiting expression of a tumor-associated gene and
expression of at least one candidate kinase gene, wherein a decrease in
tumor cell survival in the presence of inhibition of both genes compared
to the level of tumor cell survival in the presence of inhibition of
solely said tumor-associated gene indicates that said candidate kinase
gene is a tumor survival kinase.

24-26. (canceled)

Description:

FIELD OF THE INVENTION

[0001] This invention relates to compounds and methods for cancer therapy.

BACKGROUND OF THE INVENTION

[0002] The role of p53 as a tumor suppressor is generally attributed to
its ability to stop the proliferation of precancerous cells by inducing
cell-cycle arrest or apoptosis. This tumor suppressor gene is mutated in
many human cancers and results in the loss of a cell's ability to survey
for DNA damage. Inactivation or disruption of the p53 tumor suppressor
gene is a common event in the development of most types (50-80%) of human
cancers.

SUMMARY OF THE INVENTION

[0003] The present invention provides compounds and methods to
preferentially or specifically target tumor cells, e.g., inhibiting their
proliferation or decreasing their survival, while sparing normal cells.
Non-tumor cells are spared, because the compounds inhibit a kinase that
becomes necessary for survival only when the process of carcinogenesis is
initiated and remains necessary after the cell becomes cancerous.
Identification of such specific therapeutic agents was possible only
after elucidating a family of kinases that are not needed in normal cells
but are necessary for survival of tumor cells. Such kinases are
characterized or classified as tumor survival kinases. These kinases
become essential in cells in which p53 is deficient, e.g., mutated,
inactivated, or otherwise compromised or reduced. The compounds are used
to inhibit proliferation or kill p53-deficient tumors in individuals,
e.g., human patients, that have been diagnosed with a p53-deficient
tumor.

[0004] Inhibitors of these kinases are superior to many existing
anti-tumor drugs, because they preferentially act on p53-deficient tumor
cells compared to non-tumor cells or cells in which p53 expression or
activity is normal. Tumor survival kinases include serum- and
glucocorticoid-induced protein kinase (SGK) and p21-activated kinase
(PAK). For example, a method of inhibiting proliferation or decreasing
proliferation of a p53-deficient tumor cell involves contacting the tumor
cell with a composition comprising an inhibitor of SGK2, PAK3, or CDK7.
The compounds inhibit proliferation of tumor cells or precancerous cells.

[0005] In one embodiment, the compounds inhibit the enzymatic activity or
expression of a p53-dependent tumor cell survival kinase, e.g., PAK3 or
SGK2, thereby reducing cell proliferation and/or causing death of the
tumor cell. p53-deficient cells are contacted with an inhibitor of a
tumor survival kinase. The p53-deficient cell is a p53 deficient tumor
cell, a human papilloma virus (HPV)-infected cell (e.g., a non-tumor
cell), or a non-tumor cell expressing an HPV oncoprotein. p53-deficient
tumors affect many different tissue types. For example, the compounds are
administered to a subject diagnosed as suffering from or at risk of
developing a p53-deficient cell condition such as cancer or a
precancerous lesion or mass. The cell to be treated is, e.g., a tumor
cell or tumor cell line of a tissue type selected from the group
consisting of breast, cervix, uterus, bladder, brain, lung, esophagus,
liver, prostate, colon, brain (e.g., glioblastoma). Mutations associated
with p53 deficiency (decrease or absence of expression level or enzymatic
activity) is also associated a variety of sarcomas and leukemias.

[0006] In one example, the method involves contacting the cell, e.g., a
tumor cell, with a composition comprising an inhibitor of PAK3, wherein
said inhibitor comprises the structure of PAK3 inhibitor Chemotype 4

##STR00001##

Inhibitors that belong to this chemotype group include LDN-0211958,
LDN-0211959, LDN-0026056, LDN-0211955, LDN-0041012, and LDN-0028618. In
another example, cells are contacted with a composition comprising an
inhibitor of PAK3, wherein said inhibitor comprises the structure of PAK3
inhibitor Chemotype 8

##STR00002##

Exemplary compounds include LDN-0044878 or LDN-0091420.

[0007] In yet other example, the method of inhibiting proliferation of or
killing a p53-deficient cell is carried out by contacting the cell with a
composition comprising any one of the inhibitors shown in FIGS. 9A-T,
e.g., inhibitor that have general structure selected from PAK3 inhibitor
Chemotypes 1, 2, 3, 3a, 3b, 3c, 3d, 4, 5, 6, 7, 8, 8a, 9, 10, 11, 12, 13,
14. Other PAK3 inhibitory compounds useful in the these methods include
LDN-0047862, -0009460, -0042112, -0097519, -0096422, -0111371, -0086947,
-001731, -0080086, and -0097728.

[0008] The invention also includes methods of inhibiting proliferation of
or killing a p53-deficient cell by targeting tumor survival kinase, SGK2.
This method involves contacting the cell, e.g., the cell types described
above, with a composition comprising an inhibitor of SGK2 that comprises
the structure of SGK inhibitor chemotype 1A

##STR00003##

An exemplary composition comprises LDN-0149188. In another example, the
method is carried out using an inhibitor of SGK2, wherein said inhibitor
comprises the structure of SGK inhibitor chemotype 2A

##STR00004##

such as LDN-0144705 or LDN-0144676. In yet another example, the method
involves contacting cells with a composition comprising an inhibitor of
SGK2, wherein said inhibitor comprises the structure of SGK inhibitor
chemotype 4

##STR00005##

An exemplary compound that belongs to SGK inhibitor group chemotype 4 is
LDN-0169731. Other useful SGK inhibitors comprise a general structure
selected from SGK2 inhibitor Chemotypes 1, 1A, 1B, 2, 2A, and 4 as
exemplified by compounds shown in FIGS. 11-13 as well as those shown in
FIG. 14 (e.g., LDN-0181476 or LDN-0187289).

[0009] Analogues or derivatives of the aforementioned compounds are also
useful in the described methods provided that the structure or chemical
formulas comply with the general structures shown in FIG. 10 (for PAK3
inhibitor derivatives) or FIG. 15 (for SGK2 inhibitor derivatives).

[0010] A method of identifying a tumor survival kinase comprises
synthetically inhibiting expression of a tumor-associated gene and
expression of at least one candidate kinase gene. A decrease in tumor
cell survival in the presence of inhibition of both genes compared to the
level of tumor cell survival in the presence of inhibition of solely the
tumor-associated gene (e.g., p53) indicates that the candidate kinase
gene is a tumor survival kinase. For example, kinase targets are
identified by depleting p53 (or other kinases) by infection with a
lentiviral shRNA. Tumor survival kinases are identified by detecting
combinations that lead to pronounced decreased in cell viability. For
example, co-depletion of p53 and PAK3 or SGK2 resulted in a dramatic
decrease in cell proliferation/viability, whereas depletion of an
unrelated kinase, MAP3K8, lead to a similar effects in control and p53
depleted cells. This synthetic lethality approach is useful to identify
tumor survival kinases, which are useful targets for anti-tumor drugs.

[0011] A method of identifying an anti-tumor agent for inhibition of p53
deficient tumor cells, is carried out by contacting tumor survival kinase
with a candidate compound and determining whether the candidate compound
inhibits enzymatic activity of the kinase. A reduction in a level of
activity in the presence of the candidate compound compared to that in
the absence of the candidate compound indicates that the candidate
compound preferentially inhibits p53 deficient tumor cells. A method of
identifying an anti-tumor agent for inhibition of p53 deficient tumor
cells is carried out by contacting a cell dependent upon a tumor survival
kinase with a candidate compound and determining whether the candidate
compound inhibits survival or proliferation of the cell. A reduction in a
level of survival or proliferation in the presence of the candidate
compound compared to that in the absence of the candidate compound
indicates that the candidate compound preferentially inhibits p53
deficient tumor cells. Exemplary

tumor survival kinases include those described above--serum- and
glucocorticoid-induced protein kinase (SGK), a p21-activated kinase
(PAK), as well as a cyclin-dependent protein kinase (CDK) such as CDK7.

[0012] Compounds identified by such screens are useful for inhibiting
proliferation of or killing a p53-deficient cells such as tumor cells.
The compounds inhibit or decreases enzymatic activity of SGK2 or PAK3. A
reduction in a level of the activity in the presence of the candidate
compound compared to that in the absence of the candidate compound
indicates that the candidate compound preferentially inhibits p53
deficient tumor cells. For example, enzymatic activity is reduced by 20%,
50%, 75%, or more (e.g., 2-fold, 5-fold, 10-fold, or more).

[0013] A cell dependent upon a tumor survival kinase is a cancer cell,
e.g., a p53 deficient tumor cell, or a human papilloma virus
(HPV)-infected cell, or a non-tumor cell expressing an HPV oncoprotein.
The tumor cell to be treated or tumor cell line to be tested is of a
tissue type selected from the group consisting of bladder, brain, breast,
cervix, colon, esophagus, head and neck, liver, lung, pancreas, prostate,
soft tissue, stomach, uterus, leukemias and lymphomas.

[0014] In one embodiment, the compounds of the disclosure include a
heterocyclic group comprising at least two nitrogen atoms. Examples of
suitable diazaheterocycles include imidazolidine, pyrazolidine,
piperazine, pyrimidine, pyridazine, pyrazine, and annulated bicyclic
compounds comprising such diazaheterocycles. In one group of preferred
embodiments, the compounds are diaza heterocyclic compounds that further
comprises an amide moiety. The amide moiety may be part of a cyclic
portion of the compound, and/or may be part of a linear portion of the
compound.

[0015] The compounds to be used in the methods described herein are
purified. For example, the compounds are chemically synthesized and
separated from starting ingredients and by-products using known methods
such as chromatographic techniques. A purified compound comprises at
least 75%, 80%, 90% or 99%-100% by weight (w/w).

[0016] As used herein, the phrase "having the formula" or "having the
structure" is not intended to be limiting and is used in the same way
that the term "comprising" is commonly used. The term "independently
selected from" is used herein to indicate that the recited elements,
e.g., R groups or the like, can be identical or different.

[0017] As used herein, the terms "may," "optional," "optionally," or "may
optionally" mean that the subsequently described circumstance may or may
not occur, so that the description includes instances where the
circumstance occurs and instances where it does not. For example, the
phrase "optionally substituted" means that a non-hydrogen substituent may
or may not be present on a given atom, and, thus, the description
includes structures wherein a non-hydrogen substituent is present and
structures wherein a non-hydrogen substituent is not present.

The term "alkyl" as used herein refers to a branched or unbranched
saturated hydrocarbon group typically although not necessarily containing
1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as
cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Reference
to specific alkyl groups is meant to include all constitutional isomers
that exist for that group. Generally, although again not necessarily,
alkyl groups herein may contain 1 to about 18 carbon atoms, and such
groups may contain 1 to about 12 carbon atoms. The term "lower alkyl"
intends an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers
to alkyl substituted with one or more substituent groups, and the terms
"heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl
substituent in which at least one carbon atom is replaced with a
heteroatom, as described in further detail infra. If not otherwise
indicated, the terms "alkyl" and "lower alkyl" include linear, branched,
cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or
lower alkyl, respectively.

[0018] The term "alkenyl" as used herein refers to a linear, branched or
cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at
least one double bond, such as ethenyl, n-propenyl, isopropenyl,
n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl,
eicosenyl, tetracosenyl, and the like. Generally, although again not
necessarily, alkenyl groups herein may contain 2 to about 18 carbon
atoms, and for example may contain 2 to 12 carbon atoms. The term "lower
alkenyl" intends an alkenyl group of 2 to 6 carbon atoms. The term
"substituted alkenyl" refers to alkenyl substituted with one or more
substituent groups, and the terms "heteroatom-containing alkenyl" and
"heteroalkenyl" refer to alkenyl in which at least one carbon atom is
replaced with a heteroatom. If not otherwise indicated, the terms
"alkenyl" and "lower alkenyl" include linear, branched, cyclic,
unsubstituted, substituted, and/or heteroatom-containing alkenyl and
lower alkenyl, respectively.

[0019] The term "alkynyl" as used herein refers to a linear or branched
hydrocarbon group of 2 to 24 carbon atoms containing at least one triple
bond, such as ethynyl, n-propynyl, and the like. Generally, although
again not necessarily, alkynyl groups herein may contain 2 to about 18
carbon atoms, and such groups may further contain 2 to 12 carbon atoms.
The term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon atoms.
The term "substituted alkynyl" refers to alkynyl substituted with one or
more substituent groups, and the terms "heteroatom-containing alkynyl"
and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is
replaced with a heteroatom. If not otherwise indicated, the terms
"alkynyl" and "lower alkynyl" include linear, branched, unsubstituted,
substituted, and/or heteroatom-containing alkynyl and lower alkynyl,
respectively.

[0020] The term "alkoxy" as used herein intends an alkyl group bound
through a single, terminal ether linkage; that is, an "alkoxy" group may
be represented as --O-alkyl where alkyl is as defined above. A "lower
alkoxy" group intends an alkoxy group containing 1 to 6 carbon atoms, and
includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy,
t-butyloxy, etc. Substituents identified as "C1-C6 alkoxy" or
"lower alkoxy" herein may, for example, may contain 1 to 3 carbon atoms,
and as a further example, such substituents may contain 1 or 2 carbon
atoms (i.e., methoxy and ethoxy).

[0021] The term "aryl" as used herein, and unless otherwise specified,
refers to an aromatic substituent generally, although not necessarily,
containing 5 to 30 carbon atoms and containing a single aromatic ring or
multiple aromatic rings that are fused together, directly linked, or
indirectly linked (such that the different aromatic rings are bound to a
common group such as a methylene or ethylene moiety). Aryl groups may,
for example, contain 5 to 20 carbon atoms, and as a further example, aryl
groups may contain 5 to 12 carbon atoms. For example, aryl groups may
contain one aromatic ring or two fused or linked aromatic rings, e.g.,
phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone,
and the like. "Substituted aryl" refers to an aryl moiety substituted
with one or more substituent groups, and the terms "heteroatom-containing
aryl" and "heteroaryl" refer to aryl substituent, in which at least one
carbon atom is replaced with a heteroatom, as will be described in
further detail infra. If not otherwise indicated, the term "aryl"
includes unsubstituted, substituted, and/or heteroatom-containing
aromatic substituents.

[0022] The term "aralkyl" refers to an alkyl group with an aryl
substituent, and the term "alkaryl" refers to an aryl group with an alkyl
substituent, wherein "alkyl" and "aryl" are as defined above. In general,
aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms. Aralkyl
and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as
a further example, such groups may contain 6 to 12 carbon atoms. The term
"amino" is used herein to refer to the group --NZ1Z2 wherein
Z1 and Z2 are hydrogen or nonhydrogen substituents, with
nonhydrogen substituents including, for example, alkyl, aryl, alkenyl,
aralkyl, and substituted and/or heteroatom-containing variants thereof.
The terms "halo" and "halogen" are used in the conventional sense to
refer to a chloro, bromo, fluoro or iodo substituent.

[0023] The term "heteroatom-containing" as in a "heteroatom-containing
alkyl group" (also termed a "heteroalkyl" group) or a
"heteroatom-containing aryl group" (also termed a "heteroaryl" group)
refers to a molecule, linkage or substituent in which one or more carbon
atoms are replaced with an atom other than carbon, e.g., nitrogen,
oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or
sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent
that is heteroatom-containing, the term "heterocyclic" refers to a cyclic
substituent that is heteroatom-containing, the terms "heteroaryl" and
heteroaromatic" respectively refer to "aryl" and "aromatic" substituents
that are heteroatom-containing, and the like. Examples of heteroalkyl
groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated
amino alkyl, and the like. Examples of heteroaryl substituents include
pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl,
pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples
of heteroatom-containing alicyclic groups are pyrrolidino, morpholino,
piperazino, piperidino, tetrahydrofuranyl, etc.

[0024] "Hydrocarbyl" refers to univalent hydrocarbyl radicals containing 1
to about 30 carbon atoms, including 1 to about 24 carbon atoms, further
including 1 to about 18 carbon atoms, and further including about 1 to 12
carbon atoms, including linear, branched, cyclic, saturated and
unsaturated species, such as alkyl groups, alkenyl groups, aryl groups,
and the like. "Substituted hydrocarbyl" refers to hydrocarbyl substituted
with one or more substituent groups, and the term "heteroatom-containing
hydrocarbyl" refers to hydrocarbyl in which at least one carbon atom is
replaced with a heteroatom. Unless otherwise indicated, the term
"hydrocarbyl" is to be interpreted as including substituted and/or
heteroatom-containing hydrocarbyl moieties.

[0025] The term "cyclic" as used herein refers to a molecule, linkage, or
substituent, that is or includes a circular connection or atoms. Unless
otherwise indicated, the term "cyclic" includes aromatic, alicyclic,
substituted, unsubstituted, heteroatom-containing moieties, and
combinations thereof.

[0027] When the term "substituted" appears prior to a list of possible
substituted groups, it is intended that the term apply to every member of
that group. For example, the phrase "substituted alkyl and aryl" is to be
interpreted as "substituted alkyl and substituted aryl." By two moieties
being "connected" is intended to include instances wherein the two
moieties are directly bonded to each other, as well as instances wherein
a linker moiety (such as an alkylene or heteroatom) is present between
the two moieties.

[0028] Unless otherwise specified, reference to an atom is meant to
include isotopes of that atom. For example, reference to H is meant to
include 1H, 2H (i.e., D) and 3H (i.e., T), and reference
to C is meant to include 12C and all isotopes of carbon (such as
13C).

[0029] The compounds and methods described herein have numerous advantages
over existing treatments because they target tumor cells, e.g., tumor
cells in which p53 expression is deficient or lost, and spares normal
no-tumor cells or cells that are characterized by normal p53 expression.

[0030] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In the case of conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and not intended
to be limiting.

[0031] Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments thereof, and
from the claims. References cited, including the contents of GENBANK
Accession Numbers are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1 is a list showing identification of protein kinases that
become essential as a consequence of HPV oncoprotein expression in
primary human keratinocytes. HeLa, SiHa and CaSki cervical carcinoma
(CxCa), high passage (HP) and low-passage (LP) HPV16 immortalized
keratinocytes as well as human foreskin keratinocytes (HFKs) engineered
to express the entire HPV16 early coding region (ER) or E6 and/or E7
oncoproteins were infected with lentiviral vectors expressing shRNAs to
individual kinases. The percentages of the decrease in cell
proliferation/survival normalized to a scrambled control shRNA and
compared to HFKs as determined by Alamar blue assays are shown. The
numbers represent averages of 2 to 4 independent experiments, each
performed in quadruplicate. CxCa represent averages of the 3 cervical
carcinoma lines tested. Only kinases that show average differences of
≧50% (CxCa and HPV-immortalized HFKs) or ≧40% (HPV-oncogene
expressing HFK populations) are shown.

[0033] FIG. 2A is a series of line graphs showing that multiple PAK3 and
SGK2 lentiviral shRNA expression vectors inhibit proliferation/viability
of CaSki, SiHa and HeLa cervical carcinoma cells more efficiently than in
primary human foreskin keratinocytes (HFK) at multiple concentrations.
Cell proliferation/viability was assessed by Alamar blue staining

[0035] FIG. 2C is a series of photomicrographs showing that multiple PAK3
and SGK2 shRNA expression vectors inhibit proliferation/viability of
HPV16 E6 expressing HFKs more efficiently than matched control HFKs.
Cells were stained with crystal violet and photographed.

[0037] FIG. 3B is a photomicrograph, bar graph, and photograph of a
Western blot. HFKs infected with a control or p53 specific shRNA
expression vector (3756), were infected with shRNA expression vectors
encoding scrambled, SGK2, PAK3 or MAP3K8 specific shRNAs.
Photomicrographs are shown in the left. panel, a Western blot documenting
p53 depletion is shown in the middle panel and quantification of Alamar
blue assays are shown in the right panel. The data show that inhibition
of cell proliferation/viability by SGK2 and PAK3 depletion is related to
loss of p53 tumor suppressor activity.

[0038] FIG. 4A is a photomicrograph and a bar graph. Primary human mammary
epithelial cell infected with a control or p53 specific shRNA expression
vector were infected with shRNA expression vectors encoding scrambled,
SGK2 or PAK3 specific shRNAs. Photomicrographs are shown in the left
panel and quantification of Alamar blue assays are shown in the right
panel. FIG. 4B is a bar graph. Primary human prostate epithelial cells
infected with a control or p53 specific shRNA expression vector were
infected with shRNA expression encoding scrambled, SGK2 or PAK3 specific
shRNAs. Quantifications of Alamar blue assays are shown. The data show
that depletion of p53 causes synthetic lethality with SGK2 and PAK3 loss
in primary human epithelial cells derived from multiple tissues.

[0040] FIG. 6 is a photograph of a Western blot showing expression of
HPV16 E7, pRB and p53 in HFK populations. Decreases in p53 and pRB steady
state levels served a surrogate marker for HPV16 E6 or E7 expression,
respectively.

[0041] FIGS. 7A-D are tables showing the results of essential kinase
screens performed with cervical carcinoma and primary human foreskin
keratinocyte (HFK) cells. Cells infected with the indicated kinase
specific lentiviral shRNA expression vectors were assessed for cell
viability using Alamar Blue. Percent viability was normalized to cells
infected with a scrambled (SCRAM) shRNA control vector. The average
percent loss of viability determined for each cell line, calculated from
two to four independent shRNA screens each performed in quadruplicate, is
given for each shRNA expression vector tested. Ave CxCa and Ave HKF
denote the average percent loss of viability in the cervical cancer lines
and the two independent primary human foreskin keratinocyte (HFK)
populations, respectively. Percentages of difference in viability of
cervical cancer lines as compared to HFKs is also listed.

[0042] FIGS. 8A-D are tables showing the results of essential kinase
screens performed with cervical carcinoma cell lines (CxCa), late passage
(HKc/DR) and early passage (HKc/HPV16) HPV16 immortalized keratinocyte
lines and passage/donor matched keratinocyte populations expressing the
HPV16 early coding region (16ER), HPV16 E6/E7 (16E6/E7), E6 (16E6) or E7
(16E7). Cells infected with the indicated kinase specific lentiviral
shRNA expression vectors were assessed for cell viability using Alamar
Blue. Average percent viability calculated from two to four independent
shRNA screens each performed in quadruplicate was normalized to cells
infected with a scrambled (SCRAM) shRNA control vector. Percentages of
difference in viability compared to HFKs are listed for each cell
population tested.

[0060] In lung cancer, a mutagen found in cigarette smoke binds to DNA and
ultimately can cause G (guanine) to T (thymine) substitutions in DNA.
Other chemicals in cigarette smoke have been shown to produce C
(cytosine) to A (adenine) changes. When these occur in the p53 gene, the
mutations can cripple the p53 protein, disrupting its tumor-suppressing
function.

[0061] Two major causes of liver cancer are infection with the Hepatitis-B
virus and exposure to aflatoxin, a mutagen produced by a mold that grows
on improperly stored grains and food crops, specifically wet corn.
Aflatoxin, like benzopyrene, may alter the gene that encodes p53, thereby
disrupting the tumor-suppressing ability of p53. The Hepatitis-B virus
works to inactivate p53 in a different way; it produces a protein that
has the ability to bind p53 and prevent it from interacting effectively
with its target genes.

[0062] In skin cancer, ultraviolet (UV) rays in sunlight can cause damage
to DNA. If the DNA in a skin cell is damaged beyond repair, the p53
protein can induce cell death. However, if the UV light causes a mutation
in the p53 gene rendering the protein nonfunctional, the damaged cell may
reproduce and potentially lead to the formation of a cancerous growth.

[0063] HPV is a sexually transmitted virus that can infect cervical cells.
Once inside the cell, the virus produces a protein that binds to p53 and
causes the p53 protein to be degraded. The result of this degradation is
a decrease in available p53 protein and a loss of functional p53
activity.

[0064] In many breast cancers, the p53 gene appears to be normal. However,
in some cases the protein MDM2 is enhanced in the cells and binds to the
p53 protein, inhibiting its antitumor activity. This allows for the
growth of malignant breast cells and inhibits the p53 induced apoptotic
pathway.

[0065] Thus, p53 is implicated in cancers of the bladder, brain, breast,
cervix, colon, esophagus, larynx, liver, lung, ovary, pancreas, prostate,
skin, stomach, and thyroid. Among common tumors, with 60% of colorectal
cancers, 70% of lung cancers, and 40% of breast cancers carry p53
mutations. p53 is also linked to cancers of the blood and lymph nodes,
including Hodgkin's disease, T cell lymphoma, and certain kinds of
leukemia. The compositions and methods of the invention are useful to
treat the foregoing tumor types.

HPV and Carcinogenesis

[0066] Papillomaviruses are small double stranded DNA viruses. Subtypes
HPV-16 and HPV-18 cause cervical cancers. HPV viral oncoproteins E6 and
E7, transform cells and are necessary to maintain a malignant phenotype.
If E6 and E7 are removed, cervical cancer cells die. Both E6 and E7 bind
to and inactivate cellular targets such as tumor suppressor proteins p53
and retinoblastoma (Rb). The HPV model of cancer progression is well
characterized at a molecular level, with E6 and E7 expression being
causative agents at early stages of carcinogenesis. Experiments were
therefore carried out to identify kinases required at various stages of
carcinogenesis, e.g., after E6 expression, after E7 expression, after
immortalization, after transformation, and at various stages of cervical
carcinoma development.

[0067] Functional Screen for Kinase Requirements.

[0068] Loss of function screens were carried out to determine kinase
requirements in different cell lines using a lentivirus vector system
that produces shRNA targeting kinases. Loss of function shRNA screens
determined kinase requirements in human cell lines. Cells were transduced
with a lentiviral shRNA library that targeted kinase family members, and
cell lines were compared to evaluate growth inhibition. Downregulation of
the same kinase was found to have a different effect depending upon the
cell lines. For example, in one cell line, loss of a certain kinase had a
minor effect on cell growth. However, in another cell line, loss of the
same kinase was found to have a profound effect on cell growth, i.e., a
much greater growth inhibitory effect was observed. Thus, downregulation
or inhibition of the same kinase has different effects in different
cells.

[0069] Screens were conducted to identify kinases that are required for
proliferation and viability of human cells (FIGS. 23-24). A human
cervical cancer cell line (HeLa) and a renal cancer cell line (293T) were
screened using an RNAi library that targeted 88% of the kinome. The
screen identified 100 shRNA hits that inhibited growth (greater than or
equal to 50% inhibition) in either HeLa, 293T, or both cell lines (100
hits). These hits were then evaluated in 37 cell lines, including HPV
oncoprotein expressing primary cells, cervical cancer cells, renal cancer
cells in the absence and presence of the VHL tumor suppressor gene,
breast cancer cells, and matched normal control cells of different
tissues. The 100 hits represented 88 unique kinases. Some genes scored
two shRNAs (e.g., ERRB3, Pak3), and others scored three or four (e.g.,
Jnk3 scored four shRNAs. The results revealed various cell lines
downregulated for these genes showed different kinase signatures.

[0070] The essential kinase signatures were found to be remarkably
different when comparing cell lines representing various tumor types, and
similarities are detected only in particular settings. For example,
comparison of primary cells from the same tissue and of the same lineage,
irrespective of the individual donor or the date of collection yields a
very similar pattern of kinase requirements. Comparison of cells that are
identical except for the expression of a single gene, for example an
oncogene or a tumor suppressor gene, reveals distinct changes in kinase
requirements, allowing the identification of key changes in cell
metabolism that are mediated by the gene in question. Whereas most tumor
cells, even those isolated from the same site had different kinase
requirements, we discovered a limited number of examples of tumor cells
from the same site with closely related patterns of kinase requirements.
In particular, the HPV18 positive adenocarcinoma cell line HeLa and the
HPV16 positive squamous cell carcinoma line CaSki were amongst the most
closely related tumor cell lines, whereas the HPV16 positive squamous
cell carcinoma line SiHa showed a more distinct pattern of kinase
sensitivity.

[0071] In addition to the kinase profiling described above, focused
screens were conducted to identify kinases that are involved at various
stages in HPV-associated disease. The 100 hits (WO 2007/044571 A2) were
screened using HPV oncogenes (e.g., HPV-16 oncogenes) and normal foreskin
keratinocytes, normal keratinocytes expressing HPV oncoprotein E6, normal
keratinocytes expressing E7, normal keratinocytes expressing both E6 and
E7, normal keratinocytes expressing the entire early region of the virus
(E6, E7, and other proteins, and cervical cancer cells to identify shRNAs
that inhibit growth of oncoprotein expressing cells and cancer cells
compared to controls. Expression of E6 downregulates p53, E7
downregulates RB, and both together as well as the entire early region
can downregulate both p53 and RB.

[0072] CDK7, PAK3, and SGK2 shRNAs were found to be more effective at
inhibiting growth in all three oncoprotein expressing cell lines compared
to normal keratinocyte control cells. SGK2 showed the most pronounced
differential. Numerous cell lines were tested, and downregulation of
CDK7, PAK3 and SGK2 led to enhanced growth inhibition at early stages of
immortalization and at later stages of carcinoma. These results indicated
the synthetic lethal interactions exist between p53 and several protein
kinases and that loss of p53 makes cells reliant on novel kinases for
survival. p53 loss makes cells dependent on SGK2 and PAK3, e.g., primary
epithelial cells that lose p53 become dependent on SGK2 and PAK3. p53
loss changes the regulation of epithelial cells and induces the
requirement for the kinases such as SGK2 and PAK3, and cells with
non-functional p53 require the kinases SGK2 and PAK3.

SGK2

[0073] SGK2 is a serine/threonine protein kinase. Although the gene
product is similar to serum- and glucocorticoid-induced protein kinase
(SGK), this gene is not induced by serum or glucocorticoids. This gene is
induced in response loss of p53 as a result of mutation or HPV infection.

[0074] SGK kinases are members of the "AGC" subfamily (which includes
protein kinase A (PKA) protein kinase B (PKB, and protein kinase G
(PKG)), and there are three SGK isoforms. The serum- and
glucocorticoid-inducible kinase 1 (SGK1) was the first cloned, and
originally found to be an early response gene that was transcriptionally
activated by serum and glucocorticoids. SGK2 kinase is closely related
(80% homology) to SGK1 and SGK3, in addition to showing 54% homology to
protein kinase B (AKT) in its catalytic domains. The SGK kinases become
activated and function through their phosphorylation by PI 3-kinase
family members, including the 3-phosphoinositide (PIP3)-dependent kinase
PDK1. SGK1 is phosphorylated at one major site in vitro by PDK1, and SGK2
and SGK3 kinases are phosphorylated at two major sites, including a Thr
residue in the activation loop and a Ser in a hydrophobic motif. Like PKB
and SGK1, the substrate specificity of SGK2 and SGK3 involves the
phosphorylation of Ser and Thr residues that lie in
Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs. SGK1 function plays an important role
in activating potassium, sodium, and chloride ion channels, and plays a
role in regulating processes such as cell survival, neuronal
excitability, and renal sodium excretion. The SGK1 gene contains
p53-binding sites in its promoter.

PAK3

[0075] PAKs, e.g., PAK3 are also serine/threonine protein kinases. These
kinases bind to and, in some cases, are stimulated by activated forms of
the small GTPases, Cdc42 and Rac. PAK3 was also found to be induced in
response loss of p53 as a result of mutation or HPV infection.

PAK3 is a serine/threonine protein kinase that belongs to the "STE"
subfamily and there are six PAK isoforms. PAKs are key regulators of
cancer signaling pathways. PAK1 is the best characterized member and was
originally identified as a protein that interacts with CDC42 and RAC1,
which are members of the Rho GTPase family of proteins. The
GTPase-activated PAKs localize to the leading edge of cells and function
to stimulate cell motility and invasion. Increased PAK1 expression and/or
activity have been linked to several cancers including breast, colon,
ovarian, bladder, brain and T-cell lymphomas (Kumar et al., 2006, Nat.
Rev, Cancer. 459-71.). Increased PAK4 expression has been confirmed in
pancreatic cancers. All six PAK genes carry p53 consensus binding sites
in their promoters.

[0076] PAK3 inhibition is synthetically lethal in combination with
expression of a single HPV oncogene, E6, in primary human epithelial
cells. The major cellular target of E6 is p53, which is targeted for
proteasome-mediated degradation upon binding to E6. PAK3 inhibition leads
to preferential cell death in cells that have lost p53 tumor suppressor
activity.

CDK7

[0077] This protein forms a trimeric complex with cyclin H and MAT1, which
functions as a Cdk-activating kinase (CAK). It is an essential component
of the transcription factor TFIIH, that is involved in transcription
initiation and DNA repair. This protein is thought to serve as a direct
link between the regulation of transcription and the cell cycle. CDKs are
phosphorylated within the activation segment (T-loop) by a CDK-activating
kinase (CAK) to achieve full activity. As with the other kinases
described above, CDK7 is induced in response loss of p53 as a result of
mutation or HPV infection.

[0078] The following materials and methods were used to generate the data
described herein.

Methodology

[0079] Experiments were performed using several control lentiviruses
(those expressing GFP, expressing scrambled shRNAs, and expressing shRNAs
for commonly required kinases are always included in our tests), and
procedures were thoroughly assessed and validated.

[0080] Relative viral titers were tested by comparing the levels of
puromycin-N-acetyl transferase (PAC) sequences in the virus stocks. PAC
sequences in the viral stocks were found to be within two-fold of one
another and therefore not significantly variable. Additionally, a test
plate with negative control viruses from each batch is tested for
accurate viral titers of drug-resistant colonies following transduction
of test mouse cells. Parallel cultures of GFP-expressing lentiviruses
yielded similar levels of fluorescence following infection.

[0081] Single doses of lentivirus shRNAs measured at a single time-point
show differences in their responses among cell lines. To test the
differences more quantitatively multiple cell lines (HFKs, HFKs+E6 and
cervical cancer cell lines) were used in more comparative screens,
assaying for preferential killing of oncoprotein expressing cells and
cancer cells over the normal HFKs. First, a time course of shRNA
knockdown was used to study the specific kinases required for cell
proliferation. Although each targeted kinase mRNA exhibits its own decay
curve and subsequent individual protein degradation time, Day 6
post-infection was determined to be the best point to compare the effects
of shRNA expression. Second, a viral titration was performed and used to
deliver shRNAs to cells over a wide range of viral MOI's. Viral
transductions were done with different dilutions of virus supernatant.
AlamarBlue readings were made at 6 days post infection and values were
normalized to the non-killing scrambled control shRNA and converted to
percent reduction in viability.

[0082] Experiments were also performed to demonstrate that differential
effects observed between cell lines were due to specific down-regulation
of kinase mRNAs, and that down-regulation occurred irrespective of the
functional outcome. Various shRNAs homologous to different regions of
particular kinase mRNAs were used to demonstrate that similar phenotypes
are induced upon infection. Multiple shRNAs do show similar phenotypic
changes, making it unlikely that the resulting phenotypes were due to
off-target effects.

[0083] Time courses of mRNA decay were also performed, and similar decay
profiles were seen in both cell lines, despite different levels of cell
survival. These data suggest that siRNA machinery works similarly in
different cell lines. Additionally, GAPDH mRNA levels were measured
during various time-points post-infection with the lentiviruses. GAPDH
levels were found to not significantly change during infection,
illustrating that decay of kinase mRNA levels is due to siRNA action, not
a consequence of impending cell death.

[0084] In another study using the 100 shRNA hits, the most detailed
comparisons were performed between HeLa cervical carcinoma cells and
786-0 renal carcinoma cells. 15 kinases were identified showing
differential requirements in either one tumor type or the other.

[0085] CDK4, FGFR3 and PDGFR were identified using 293T and HeLa cells.
The kinases found from these studies comprise established kinase targets,
which have been advanced as preclinical and clinical candidates for the
treatment of cancer such as CDK4, FGFR3, PDGFRB as well as previously
unknown kinase targets. These studies demonstrate the power of these
comparative screens, and methods of identifying novel therapeutic targets
for cancer.

Cell Culture

[0086] Normal human foreskin keratinocytes (HFKs) were obtained from
neonatal foreskins and cultured using standard methods. HPV oncogene
expressing cell populations were generated by transfection of appropriate
β-actin expression plasmids using nucleofection (AMAXA). HPV16 E7
expression was assessed by Western blotting; decreased p53 expression was
used as a surrogate marker for HPV16 E6 expression. In some experiments,
HPV16 E6I128T mutant was used. HFKs with p53 knockdown were obtained by
infection with appropriate lentiviral shRNA vectors followed by selection
in 2 μg/ml puromycin. Experiments were performed with donor/passage
matched cells. Low (HKc/HPV16) and high passage (HKc/DR) HPV16
immortalized cells were grown in K-SFM (Gibco). HeLa, CaSki, and SiHa
cells were grown in DMEM supplemented with 1% penicillin-streptomycin and
10% calf serum. Primary human mammary and prostate epithelial cells were
purchased from Clonetics/Lonza and grown in the specific media supplied.

Infections with shRNA Expressing Lentiviruses

[0087] Lentiviruses expressing shRNAs were produced as previously
described (40). 2,000-3,000 cells were seeded per well in 96-well plates.
Cells were infected at 24 hours after plating. Viability assays using
Alamar blue were performed after puromycin selection at five days
post-infection. Cells were stained with crystal violet for image
acquisition.

[0089] Three days post-infection with appropriate lentiviruses, cells
washed with PBS, fixed for 15 minutes with 4% paraformaldehyde in PBS,
permeabilized for 15 minutes with 0.2% Triton-X-100 in PBS and incubated
for 2 hours with either LC3 rabbit polyclonal antibody (Santa Cruz
Biotechnology) or Cleaved Caspase-3 rabbit polyclonal antibody (Cell
Signaling Technology) diluted in blocking buffer consisting of 0.5% BSA
in PBS. The secondary antibody was a goat anti-rabbit antibody conjugated
with Alexa Fluor 488 (Molecular Probes/Invitrogen) and the final two-hour
incubation step also contained rhodamine, phalloidin and Hoechst 33258
dyes (Molecular Probes/Invitrogen). Fluorescent images were acquired with
an inverted fluorescence microscope (Zeiss) at a magnification of
200×.

Reagents, Substrates and Compound Library

[0090] Recombinant human full length PAK3 enzyme was obtained
(Invitrogen), immediately aliquoted and kept in storage buffer as
purchased at -80° C. for long term storage. The HTRF®
KinEASE® kit (CisBio) containing 5× stock solution of the
enzymatic buffer and 1× detection buffer, STK substrate 2-biotin
(S2), a phosphospecific monoclonal Europium-labeled Cryptate antibody,
which recognizes a phospholylation epitope of the biotinylated peptide,
as donor fluorophore and Stretptavadin-linked XL665 (SA-XL665)
representing the acceptor fluorophor were used for TR-FRET assays. The
compound library consisted of approximately 125,000 small molecules,
including compounds approved by the Food and Drug Administration (FDA), a
purified natural products library and commercially available compounds
from various vendors. All small molecules generally adhere to Lipinski's
rules and have been optimized for maximization of molecular diversity.

Enzyme Based Kinase Assays and Hit Identification

[0091] An enzyme based kinase assay was carried out using full length
recombinant PAK3 kinase. A model biotinylated peptide RRRSLLE (SEQ ID
NO:5) was used as the substrate. Detection was done by Homogeneous Time
Resolve Fluorescence (HTRF) with an antibody that recognizes the
phosphorylation site on the peptide.

[0092] Approximately 150,000 compounds were screened using this PAK3
assay. Compounds were assayed at 10 microM and a percent inhibition of
the enzyme by the compounds were determined. Those compounds that
inhibited PAK3 by >50% at 10 microM were identified as hits. The PAK3
inhibitors identified in that matter as well as derivatives thereof
(which possess the same or similar kinase inhibitory activity) cause
preferential cell death in cells that have lost p53 tumor suppressor
activity and are therefore useful as anti-cancer agents.

[0093] TR-FRET assays were carried out as follows. A CRS CataLyst Express
robotic arm and a Cybi-well 384 channel simultaneous pipettor were used
to carry out the High-Throughput Screen. Kinase reactions were performed
in 50 mM HEPES, pH 7.0, 0.02% NaN3, 2 mM MgCl2, 0.01% BSA, 0.1
mM Orthonanadate, 1 mM DTT, 0.001% Tween-20, 0.001% Brij-35 using
ProxiPlate-384 Plus white assay plates. In the final HTS conditions, 3.2
nM PAK3 enzyme (1.6 nM final concentration in assay) and 0.2 μM
biotinylated S2 peptide (0.1 μM peptide final, at the Km) in a
volume of 5 ml kinase buffer were added to dried solutions of 10 μM
compound and pre-incubated for 30 minutes. The kinase reaction was
initiated by addition of 5 ml of 50 mMATP per reaction (25 μM ATP
final, at the Km). The reaction was incubated for 30 minutes at room
temperature. Reaction was terminated by addition of 10 μl of a
premixed solution of EDTA, Europium cryptate-labeled antibody and
fluorophore-conjugated streptavadin. After overnight incubation at room
temperature, TR-FRET measurements were performed using a PHERAstar HTS
microplate reader, and were expressed as ratios of acceptor fluorescence
at 665 nm over donor fluorescence at 620 nm.

Cell Based Assays

[0094] HeLa cells (ATCC) were maintained in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum and 1%
Penicillin/Streptomycin. Approximately 200 to 500 cells per well were
seeded in a 96 well tissue culture plates. Following 24 h growth in
normal DMEM, the individual compounds were warmed up to room temperature
and diluted in DMEM to their according concentration and then immediately
added to the wells. One column on each assay plate contained DMEM only
("neg. control") and one column contained untreated HeLa cells as "pos.
control". Cells were treated with the compounds at various cell densities
and for treatment periods ranging between 75 hrs and approx. 200 hrs. To
control for cell viability, we assayed plates using AlamarBlue°
(Invitrogen), which uses a redox reaction to measure metabolically active
viable cells. At each time point the culture media was removed and a
solution of 10% AlamarBlue reagent and DMEM was added to each well to
measure cell viability. Fluorescence was measured after 1-2 hrs
incubation at 37 C on a Victor2 Plate Reader (PerkinElmer).

Data Analysis

[0095] Data were analyzed using GraphPad Prism Version 5 (GraphPad
Software Inc., La Jolla, Calif., USA). Each compound plate included one
column of negative controls, were no enzyme was added, and another column
for positive controls when no inhibitor compound was added; these were
used to calculate Z' factors and signal to noise ratios throughout the
screen. Percentage of inhibition of PAK3 enzyme activity was calculated
according to the following equation: % inhibition=100×(average of
positive controls-test compound value)/(average of positive
control-average of negative controls). IC50 determinations were done
in quadruplicate for each compound using different adding sequences for
compound and enzyme-substrate-mix. EC50 determinations were done in
triplicate for each compound using different time points. For calculation
of IC50 and EC50 concentrations, respectively, mean inhibition
dose response curves were fitted to the sigmoidal response equation:
Y=Bottom+(Top-Bottom)/(1+10 ((X-LogIC50))) were X is log(compound
concentration) and Y is % inhibition, and Bottom and Top are the lower
and upper plateau. Km concentrations were determined by non-linear
regression curve fitting, using the equation: Y=Vmax*X/(Km+X), where X is
the substrate concentration.

Essential Kinase Screens In Cervical Cancer Cells

[0096] To test the viability of using existing cervical cancer cell lines
to identify essential kinases, C33A (HPV negative), CaSki (HPV16 positive
at high copy number), SiHa (HPV16 positive at low copy number), and HeLa
(HPV18 positive) cells were screened in a 96-well format. For each cell
type, each well of a 96-well plate was infected with a lentiviral shRNA
expression vector that knocked-down expression of an individual kinase.
Cells were selected with puromycin to demonstrate the efficiency of the
infection. Six days post infection, alamar blue was added to the culture
media. Similar to tetrazolium salts, alamar blue measures the
mitochondrial fitness of cells. This provides a quick, convenient,
colorimetric read-out of cell number. A pink color in the well
corresponds to higher cell confluency/cell survival, while a blue color
corresponds to lower cell confluency/cell death. A range of colors
in-between pink and blue can be measured in plate reader and gives the
range of killing or arrest by individual kinase knock-downs. Screens were
carried out using the "top 100 hits" against 88 individual kinases
(identified in the previous screens described above). After screening 4
cervical carcinoma cell lines, screens were expanded to include normal
primary human foreskin keratinocytes (HFKs, 8 different populations) and
fibroblasts (2 different populations, HFFs). These screens were performed
to identify essential kinases in cervical cancers versus normal primary
human cells. Since HPV is such a unique and informative model system to
study carcinogenesis, the screens were expanded to include HFKs and cell
lines expressing the HPV16 oncoproteins. By expressing the oncoproteins,
early targeting of host kinases was determined. Numerous cell lines and
populations were tested (25 total including multiple populations of the
following control HFKs, E6 expressing HFKs, E7 expressing HFKs, E6 and E7
expressing HFKs, early region expressing HFKs, control RKO colon cancer
cells, RKOs expressing E7, control NOK (normal oral keratinocytes), NOKs
expressing E7, and control HFFs).

[0097] The range of cell survival/death and efficiency of the lentiviral
infection for each screen performed using the above cell lines was
analyzed by plotting the average Alamar blue values from each run. Each
cell line was screened in at least two independent experiments, each done
in duplicate with +/-puromycin selection. Hence, each experiment was
effectively done in quadruplicate. Every point on the graph represents
the value of each average alamar blue value and directly corresponds to
the level of cell death (for example, a blue, empty well would likely
have a reading from 1,000-10,000, whereas a pink, confluent well would
have a reading of 30,000-50,000 relative fluorescent units). All data
points generated from each viral shRNA transduction should cluster on a
linear axis, in a +puromycin (X-axis) and -puromycin (Y-axis)
scatterplot, indicating that viral transduction was approaching 100%. All
HPV positive cervical cancer cell lines infected well as determined by
the scatterplot and showed varying levels of cell survival upon
particular kinase knockdown. Other cell lines were tested, including
primary cells, and showed similar plots indicating that they were also
amenable to kinase screens.

[0098] Studies were carried out to determine whether ectopic expression of
a single viral oncoprotein in a specific cellular background would alter
kinase sensitivity. Cell lines expressing HPV16 E7 were screened,
including a colorectal carcinoma cell line (RKO) and normal, hTert
immortalized oral keratinocytes (NOKs). These screens showed differential
killing in response to several kinase knock-downs with E7 expression.
Several of the kinases identified in these E7-based screens fit the known
role of E7 to inhibit the retinoblastoma tumor suppressor protein. For
example, CDK6, a kinase that phosphorylates and inactives pRB, was
essential in cells without E7. When E7 was present, the shRNAs for CDK6
had no affect.

[0099] Additional experiments were done to identify kinases targeted by
particular oncogenes in normal, primary human foreskin keratinocytes
(HFKs). The HPV16 oncogenes were expressed individually, together and in
the context of the entire early region in normal, primary HFKs. Control
populations expressing the empty vector were also generated. Two
populations of HFKs were transfected, and cells with stable expression of
HPV genes were made by G418 selection. These cells were screened at
passage 5, prior to immortalization. Performing the screens in this
timescale gave rise to perfectly paired control cells for comparison. The
full collection of tested cells permitted interrogation at several of
stages of cervical cancer development starting from normal primary
cultures, to HPV expressing cells, immortalized HPV expressing cells,
tumorigenic HPV-expressing cells, and the cell lines isolated from
HPV-associated carcinomas. These cells were used to identify kinases that
become required as cells progress through these stages of tumor
development.

[0100] Percent killing was calculated for all cell lines screened by
normalizing alamar blue values to those with the scrambled shRNA control.
Analysis of percent killing was performed, and the shRNAs against kinases
demonstrating the largest percentage difference in killing between
cervical cancer cell lines and normal cells were determined. Kinase
knockdowns leading to a high percent of cell death in cervical cancer
cells but demonstrating a low percentage of cell death in normal cells
are of the utmost interest for the development of therapeutic targets, as
they are the most likely to be effective at killing tumor cells without
harming normal cells. Several targets identified in the screens were also
identified as essential kinases in other tumor cell lines tested.

[0101] The patterns that have emerged from these indicate that 3 kinases,
CDK7, PAK3, and SGK2 were required for proliferation of cervical
carcinoma cells, but not in normal primary keratinocytes. They become
required in cells in all stages past the expression of HPV early
proteins, and expression of E6 alone is both necessary and sufficient to
establish their need in cells. Although the data indicate that CDK7 is a
member of this class of kinases, the strength of its response was less
than that of SGK2 and PAK3.

[0102] The action of HPV E6 proteins changes cell metabolism in such a way
as to make keratinocytes now require the action of these kinases. All
three cervical carcinoma cells tested rely on the independent action of
these kinases, and multiple populations of primary keratinocytes
development dependence on these kinases following E6 expression.
Surprisingly, the inhibition of p53 by the HPV E6 protein induced
dependence on SGK2 and PAK3. This observation has been borne out by
further experimentation. SGK2 and PAK3 knockdowns using multiple shRNAs
for each kinase and in repeated experiments had no effect on the fate or
rate of proliferation of primary keratinocytes. However, the expression
of HPV E6 but not mutations of E6 that fail to degrade p53 induced
dependence on SGK2 or PAK3 in primary keratinocytes. Further, loss of p53
by either of two shRNAs induced dependence on SGK2 or PAK3. Finally,
expression of a dominant negative version of p53 that functionally
inactivates this protein similarly induced SGK2 and PAK3 requirements.
These affected cells die by either apoptosis or autophagy. This
phenomenon was not restricted to keratinocytes; primary mammary
epithelial cells, prostate epithelial cells, and foreskin fibroblasts
responded similarly. SGK2 and PAK3 mRNAs are also lowered dramatically by
their cognate shRNAs.

[0103] These data establish a clear genetic interaction between p53 loss
and either SGK2 or PAK3 loss. p53, SGK2, or PAK3 alone can be removed in
multiple primary (normal) cell cultures with no apparent effects.
However, the combination of p53 and SGK2 loss or the combination of p53
and PAK3 loss leads to cell death. These interactions then are
synthetically lethal. These relationships are induced by cancer
mutations, and are exploited as described herein to identify cancer
targets and therapeutic agents that inhibit those targets to kill or
decrease the proliferation of tumor cells with little or no adverse
effect on normal non-tumor cells.

Synthetic Lethal Interactions Between p53 and the Protein Kinases SGK2 and
PAK3

[0104] Studies were carried out to determine how kinase requirements
change during tumor development. SGK2 and PAK3 become essential for cell
proliferation/viability as primary epithelial cells loose p53 tumor
suppressor activity. Since loss of p53 tumor suppressor activity is the
most common hallmark of human tumorigenesis, the identification of these
kinases represent a unique class of chemotherapeutic targets--proteins
that become essential following cancer mutations that may not themselves
be mutated directly.

Kinases that are Essential for Proliferation/Survival of HPV-Positive
Human Cervical Cancer Cell Lines

[0105] Experiments were carried out to determine whether there was a
common set of kinases that were essential for proliferation/survival of
three cervical carcinoma cell lines but were dispensable for primary
human foreskin keratinocytes (HFKs). Cells were infected with the
appropriate lentiviral shRNA expression vectors, and cell
proliferation/survival was assessed by Alamar blue staining Alamar blue
is a redox-sensitive dye that interrogates mitochondrial fitness of
cells, and these assays provide a readout for cell
proliferation/viability. The raw values were normalized to a scrambled
control shRNA and are presented as % decrease in proliferation/viability.
Kinases were designated "essential" (1) when an shRNA inhibited cell
proliferation/viability ≧50% on average in the three cervical
cancer lines, and (2) when the shRNA scored as ≧50% more effective
in suppressing proliferation/viability as compared to the average
response in two populations of HFKs. From the tested set of 86 kinases
plus controls, 26 kinases (represented by 27 shRNAs) were identified that
scored as essential by these criteria (FIG. 1, FIGS. 7A-D).

Human Kinases that Become Essential at Distinct Stages of HPV-Mediated
Human Cervical Carcinogenesis

[0106] HPV-associated carcinogenesis is readily modeled in vitro using an
art-recognized model system. Thus, two HPV16-immortalized HFK lines that
model different stages of cervical carcinogenesis were evaluated. The two
cell lines, HKc/HPV16 and HKc/DR are derived from a single piece of
foreskin epithelium that was transfected with a head-to-tail dimer of the
cloned HPV16 genome. Low passage cells (HKc/HPV16) represent freshly
immortalized cells, whereas high passage cells (HKc/DR) have been
selected for resistance to differentiation and failure to growth arrest
in response to TGF-β. While both cell lines are non-tumorigenic,
mRNA expression profiling studies have shown that HKc/DR are more similar
to cervical carcinoma cells than HKc/HPV16 cells. As in the experiments
with cervical cancer lines, kinases were identified as "essential" when
their depletion yielded ≧50% difference in proliferation/survival
relative to HFKs. A total of 18 essential kinases were identified for
HKc/DR. Six of these, CDK7, HERS, JNK3, MELK, PAK3 and SGK2, were also
essential for cervical carcinoma lines. For HKc/HPV16, 27 essential
kinases were identified. Ten of these, CDK7, EPHB1, HER3, JNK3, KHS1,
MELK, MYO3B, PAK3, ROS and SGK2, were also essential for cervical cancer
lines. Seventeen of the 18 essential kinases for HKc/DR also scored as
essential in HKc/HPV16. Six of these 17 kinases, CDK7, HER3, JNK3, MELK,
PAK3 and SGK2 were essential for HKc/HPV16, HKC/DR as well as the
cervical carcinoma cell lines (FIG. 1 and FIGS. 8A-D).

Human Kinases that Become Essential as a Direct Consequence of HPV
Oncogene Expression

[0107] To identify kinases that become essential as a direct consequence
of HPV16 gene expression, two independent sets of donor/passage matched
HFK populations engineered to express the HPV16 early region or the HPV16
E6 and/or E7 oncogenes were analyzed. Expression of HPV16 E7, pRB and p53
in the corresponding HFK populations was assessed by Western blotting.
Decreases in p53 and pRB steady state levels served a surrogate marker
for HPV16 E6 or E7 expression, respectively (FIG. 6). Each of these HFK
populations was transduced with the collection of 100 shRNAs as above.
Kinases were classified as "essential" when they showed ≧40%
decreased proliferation/viability relative to normal cells in each
matched set. Six kinases (ADCK4, BTK, HUNK, PAK3, ROS, SGK2) met these
criteria in HPV16 early region expressing HFKs, 1 (SGK2) in HPV16 E6/E7
expressing HFKs, 3 (PAK3, SGK2, SURTK106) in HPV16 E6 expressing HFKs and
none scored in HPV16 E7 expressing HFKs. Whereas BTK also scored in
HKc/HPV16, and ROS in HKc/HPV16 as well as cervical carcinoma cells, only
PAK3 and SGK2 consistently scored as essential in HPV16 E6, early region
expressing HFKs, HKc/HPV16 and HKc/DR as well as in the cervical
carcinoma cell lines. These results demonstrate that HPV16 E6 expression
in primary HFKs induces synthetic lethality upon loss of SGK2 and PAK3
expression, and this is retained in HFKs expressing the entire HPV16
early region, HPV16-immortalized HFKs and cervical carcinoma lines.

shRNA Targeting of SGK2 and PAK3

[0108] To establish quantitative comparisons of the SGK2 and PAK3
responses and determine whether additional shRNAs specific for each of
the kinases yielded similar results, 4 different shRNA expressing
lentiviruses for each of the 2 kinases were tested in titration
experiments using CaSki, SiHa and HeLa cervical carcinoma cells and HFKs.
These experiments are necessary, since infection with a single dose of an
shRNA expressing lentivirus affords limited resolution, as it may not be
within the linear range of the assay. These experiments revealed that
multiple SGK2 and PAK3 specific shRNAs and at a variety of titers
inhibited proliferation/viability in each of the cervical carcinoma lines
but not in HFKs (FIG. 2A).

[0109] To confirm kinase knockdown, CaSki cells were transfected with
multiple PAK3 and SGK2 specific shRNA expression vectors. Since these
kinases are expressed in CaSki cells below the limit of detection by
Western blotting with commercially available antibodies, mRNA levels were
analyzed by quantitative reverse transcription PCR at 30 hours post
infection. These experiments demonstrated significant knockdown of PAK3
and SGK2 with each of the corresponding shRNAs (FIG. 2B).

[0110] The ability of multiple PAK3 and SGK2 specific shRNAs to suppress
proliferation/viability of HPV16 E6 expressing HFKs as compared to
matched control HFKs was also analyzed. As shown in FIG. 2C, multiple
shRNAs that target different regions of SGK2 or PAK3 mRNA inhibited cell
proliferation/survival of HPV16 E6 expressing cells but did not markedly
affect control HFKs.

Synthetic Lethality Induced by SGK2 or Pak3 Depletion in HPV16 E6
Expressing Cells is a Consequence of p53 Inactivation

[0112] The best-known cellular target of HPV16 E6 is the p53 tumor
suppressor protein. HPV16 E6 associates with the cellular ubiquitin
ligase E6AP, and the E6/E6AP complex associates with p53 and targets it
for proteasomal degradation. To determine whether the observed
sensitization of HPV16 E6 expressing HFKs was due to p53 degradation,
HFKs expressing HPV16 E6 or an HPV16 E6 I1128T mutant were generated.
These cells are defective for association with the E6AP ubiquitin ligase
and thus p53 degradation. Donor/passage matched vector transduced HFKs
were used as controls. SGK2 and PAK3 depletion markedly inhibited cell
proliferation/survival of wild type HPV16 E6 expressing cells, whereas
HFKs expressing the HPV16 E6 I128T mutant were less sensitive to SGK2 or
PAK3 depletion (FIG. 3A).

[0113] To directly assess the involvement of p53, p53 in HFKs was depleted
by infection with a lentiviral shRNA. Co-depletion of p53 and PAK3 or
SGK2 resulted in a dramatic decrease in cell proliferation/viability,
whereas depletion of an unrelated kinase, MAP3K8, which does not score as
synthetic lethal with HPV16 E6 expression, had similar effects in control
and p53 depleted HFKs (FIG. 3B).

[0114] To determine whether the observed effect was specific to human
foreskin derived keratinocytes or could be seen in primary epithelial
cell cultures derived from other human tissues, p53 was depleted in
primary human mammary and prostate epithelial cells. Similar to what was
observed in the primary HFKs, p53 loss caused synthetic lethality with
SGK2 and PAK3 depletion in mammary (FIG. 4A) and prostate epithelial
cells (FIG. 4B). These data confirm that functional inactivation of p53
induces cellular changes that render the SGK2 and PAK3 kinases essential
in primary human epithelial cells.

Mechanisms of Synthetic Lethality

[0115] The data described herein document a block to
proliferation/survival in p53-deficient cells upon depletion of PAK3 and
SGK2, further studies were carried out to investigate the mechanism of
action. A decrease in cell number as a consequence of kinase knockdowns
may result from apoptosis, autophagy, senescence or cell cycle block.
Hence, immunofluorescence experiments were carried out with antibodies
for cleaved, activated caspase 3, a marker of apoptosis, and LC3, a
marker of autophagy, in HeLa cells with knockdown of SGK2 or PAK3. Cells
were counterstained with Hoechst and phalloidin to visualize nuclei and
actin microfilaments, respectively. The data indicated that the
mechanisms of synthetic lethality in HeLa cells were different for the
two kinases; SGK2 depletion caused autophagy whereas PAK3 knockdown
resulted in caspase 3 activation, suggestive of apoptosis. Moreover, PAK3
depletion causes marked disruption of actin filament staining, indicative
of a collapse of the actin cytoskeleton, whereas no such effect was
observed with SGK2 depletion (FIG. 5).

Kinase Requirements of p53-Deficient Cells

[0116] Synthetic lethal screens are one example of a larger group of
genetic tests in which two genes can be shown to coordinately modify a
particular phenotype and thus must have related functions within an
organism. The terms "synthetic lethal" and "synthetic lethalities" were
coined in 1946 by T. G. Dobzhanzky (Dobzhanzky, T., 1946, Genetics of
Natural Populations. XIII. Recombination and Variability in Populations
of Drosophila Pseudoobscura. Genetics 31:269-90.16). In the simplest
terms, synthetic lethality is scored when either of two mutations in
different genes has no effect on their own but in combination they have a
lethal phenotype. Two logical premises have been proposed to explain how
synthetic lethality can be achieved. In one explanation, two pathways
perform redundant roles and loss of either pathway alone has no effect on
the cell phenotype. However, combining the two mutations leads to a
lethal phenotype by removing both pathways and depriving a cell of an
essential function. In the second explanation, one protein acts upstream
of the second, and loss of either has no effect. One mutation occurs in a
positively acting step and the other in a negative one. Since the two
proteins functionally balance one another, losing one will tip the
balance slightly, but losing both is catastrophic.

[0117] Two new synthetic interactions with loss of p53 tumor suppressor
activity using a limited shRNA screen were identified. The loss of SGK2
or PAK3 was lethal only when coupled to the loss of p53. The synthetic
interactions between p53 and SGK2 or between p53 and PAK3 have been
confirmed by several criteria. Loss of p53 through two methods;
expression of the HPV E6 protein and p53 depletion cooperate with SGK2 or
PAK3 loss to generate cell death. Depletion of the corresponding shRNA
targets was confirmed by at the level of mRNA expression. The synthetic
interactions between p53 and SGK2 loss or between p53 and PAK3 loss are
not limited to foreskin keratinocytes but are seen in primary human
epithelial cells from mammary or prostate tissues. The synthetic
relationship between p53 and SGK2 or PAK3 is not unique to a limited cell
type but is broadly applicable.

[0118] SGK2 depletion in p53 null cells leads to reduction in cell
proliferation/survival via autophagy, while PAK3 depletion in p53 null
cells causes apoptosis. This observation indicates that SGK2 and PAK3 are
components of two independent signaling pathways that become essential
following p53 loss.

[0119] Inhibitors that kill cancer cells by blocking the roles of proteins
such as SGK2 or PAK3 in a p53-dependent manner but spare normal cells are
useful as cancer therapeutic agents. Thus, studies were carried out to
identify such agents.

Identification of Small Molecule Inhibitors of SGK2 and PAK3

[0120] Compounds were screened using standard in vitro kinases assay.
Compounds identified in this screen were further tested using HPV16
oncoprotein (e.g., E6) expressing cells compared to normal controls.
Compounds that inhibited enzymatic activity in vitro were found to
inhibit proliferation of p53-deficient cells.

[0121] A library of compounds was screened for inhibitors of SGK2 and PAK3
activity by assaying phosphorylation of a generic peptide substrate
either directly; or indirectly by inhibiting upstream kinase PDK1 from
activating the enzyme in vitro. In both cases, the phosphorylated
substrate was detected using a specific anti-phospho peptide antibody
that is coupled with Eu3+ Cryptate and XL665 conjugated with
streptavidin. The initial screening concentration started at 20 μM,
and the ATP concentrations were varied to determine if these inhibitors
were competitive with ATP. All initial hits were re-assayed as a dose
response series with eight 3-fold dilutions and resulting final
concentrations ranged from 0.9 nM to 20 μM. Several hits emerged from
the screen. These compounds all showed initial kinase inhibition and were
dose responsive. Several hits displayed activity cell-based assays.

[0122] All of the small molecules (kinase inhibitory compounds) identified
in the screens and described herein are synthesized using methods and
reagents well known in the art of synthetic chemistry. FIGS. 16 to 21
show general synthetic schemes for the synthesis of exemplary SGK2
chemotypes. Several approaches exist for the synthesis of each chemotype.
The following synthetic examples are meant to illustrate the general
approach only, rather be an exhaustive synthetic search. For example,
Scheme 1 outlines an approach to the synthesis of LDN-0161044. This
compound and analogs can be constructed by a multi-component coupling
reaction in two steps, using amines, aldehydes and hydrazine as diversity
elements. Methods for the synthesis of such scaffolds are well known in
the art, e.g., Zhurnal Organicheskoi Khimii, 1998, 22(8), 1749.

[0123] Scheme 2 shows the synthesis of LDN-0146980 and analogs. The
synthesis is a two step process. The first step is a Suzuki reaction of
the heteroaryl bromide scaffold with variety of boronic acids in the
presence of palladium (0) catalyst. The second step is a copper acetate
mediated coupling of a heteroaryl amine with boronic acids. Both
reactions are well established transformations and variety of analogs are
prepared easily.

[0124] Scheme 3 describes the synthesis of LDN-0172996 and analogs. The
first step of the process is a displacement of an aryl bromide by an
amine nucleophile. The same transformation is accomplished using
palladium catalyzed aryl amination chemistry. Subsequent deprotection of
the aryl amine and reaction with sulfonyl chloride results in the
formation of the product. These reactions are well described in the
literature and many analogs are prepared in an efficient manner.

[0125] Scheme 4 outlines the synthesis of LDN-0180043 and analogs. The
first step is an alkylaton of an aryl amine with a bromo (or other
suitable electrophile). This step is followed by deprotection and
coupling with variety of amines to give the product scaffold. These
reactions are well established and many analogs are prepared easily.

[0126] Scheme 5 presents the synthesis of LDN-0179218 and analogs. The
process is a two step multi-component coupling reaction with aldehydes
and amines as the diversity elements. General methods for the synthesis
of this scaffold are well known in the art, e.g, in Archiv de Pharmazie,
1995, 328(2), 169.

[0127] Scheme 6 outlines the synthesis of LDN-0144707 and analogs and
presents two general approaches. Approach (a) is a multi-component
coupling reaction, based on imine formation and Diels-Alder cyclization.
The diversity elements are amines, aldehydes and dienes. The process is
catalyzed by Lewis acids, such as ytterbium triflate (Yb(OTf)3. The
second approach (b) is based on two discrete steps: first, the formation
of an imine from the reacting aldehyde and amine and second, Diels-Alder
cyclization of the imine with a variety of dienes. The reaction
transformations in both approaches are well established and many analogs
are prepared in an efficient manner.

Small Molecule Inhibitors of PAK3

[0128] A medicinal chemistry evaluation of the compounds from the screen
was carried out The compounds segregate into clusters and chemotypes. In
addition, generic Markush structures for analogs have been defined.

[0129] Analysis of the 130 structures included in the PAK3 data set
revealed chemotypes shown in FIGS. 9A-T. In all, 14 chemotypes and 10
singletons were discovered. Most of the chemotypes are distinct, although
there some overlap exists in some of the groupings. One of the chemotypes
which is present in several subtypes is based on the flavone or
isoflavone ring system. Generic chemotype structures are represented, as
well as, specific cores which exist within the generic structures.

[0130] Chemotypes 1 and 2 are simple aromatic compounds. Chemotypes 3 are
flavones. Chemotypes 6, 7, and 11 are flat poly aromatic compounds.
Chemotypes 4 and 5 have several points of diversity and a linker, which
can be varied to increase diversity. Chemotypes 8 and 8a have three aryl
groups and three linkers, which can be varied independently to produce a
large amount of variability. Chemotypes 12 and 13 also offer several
points of diversity and linkers. Methods for synthesis of these compound
is known in the art. FIG. 10 shows general structures for derivatives or
analogs of PAK3 inhibitory compounds, grouped by chemotype.

[0131] Compounds were tested in a cell-based assay (HeLa cells). The
results are summarized in the table below. IC50 and EC50 are expressed in
micromolar units.

[0132] A medicinal chemistry evaluation of the SGK2 inhibitory compounds
identified from the screen was also carried out. Clustera and chemotypes
in the structures were identified. In addition, generic Markush
structures, as well as, specific analogs are described for each
chemotype.

[0133] Upon analysis of the 22 structures included in the SKG2 data set,
several general chemotypes emerged (FIGS. 11-14). Some of the chemotypes
show structural overlap and as such, the overlapping chemotypes are
represented as subsets of the parent chemotype. Generic chemotype
structures are represented, as well as, specific cores which exist within
the generic. FIG. 15 shows general structures for derivatives or analogs
of SGK2 inhibitory compounds, grouped by chemotype.

[0134] Several compounds segregate into Chemotype 1. Chemotype 1 is
characterized by a fused ring and the two pendant aryl groups. Chemotype
2 is constructed of an aryl alkyl sulfone moiety.

[0135] Characterization of SGK2 inhibitory compounds (inhibition of kinase
activity) is summarized in the table below.

[0136] Therapeutic methods are carried out by administering pharmaceutical
formulations comprising kinase inhibitory compounds. The compounds are
administered to subjects (e.g., human patients, companion animals such as
dogs and cats, livestock such as cattle, sheep, goats, horses) that have
been determined to be suffering from or at risk of developing a
p53-deficient tumor. A reduction (deficiency) in p53 expression or a loss
of p53 expression in a cell or tissue is determined by detecting the p53
gene product (e.g., using a p53-specific monoclonal antibody) or by
measuring p53 nucleic acid (e.g., transcripts) in a cell or tissue sample
such as a tumor biopsy specimen.

[0137] Routes of administration, include, but are not limited to, oral,
rectal, topical, intravenous, parenteral (including, but not limited to,
intramuscular, intravenous), ocular (ophthalmic), transdermal, inhalative
(including, but not limited to, pulmonary, aerosol inhalation), nasal,
sublingual, subcutaneous or intraarticular delivery. Although the most
suitable route in any given case will depend on the nature and severity
of the conditions being treated and on the nature of the active
ingredient. The compounds are formulated in unit dosage form and prepared
using methods well-known in the art of pharmacy.

[0138] A pharmaceutical composition or medicament containing the inhibitor
or a mixture of inhibitors is administered to a patient at a
therapeutically effective dose to prevent, treat, or control cancer. The
pharmaceutical composition or medicament is administered to a patient in
an amount sufficient to elicit an effective therapeutic response in the
patient. An effective therapeutic response is a response that at least
partially arrests or slows the symptoms or complications of the disease.
An amount adequate to accomplish this is defined as "therapeutically
effective dose."

[0139] The dosage of active small molecule compound administered is
dependent on the species of warm-blooded animal (mammal), the body
weight, age, individual condition, surface area of the area to be treated
and on the form of administration. The size of the dose also is
determined by the existence, nature, and extent of any adverse effects
that accompany the administration of a particular small molecule compound
in a particular subject. A unit dosage for oral administration to a
mammal of about 50 to 70 kg may contain between about 5 and 500 mg of the
active ingredient. Typically, a dosage of the active small molecule
compound of the present invention, is a dosage that is sufficient to
achieve a therapeutic effect, e.g., reduced proliferation of tumor cells,
death of tumor cells, and/or reduction in tumor burden or tumor mass.

[0140] Optimal dosing schedules can be calculated from measurements of
small molecule compound accumulation in the body of a subject. In
general, dosage is from 1 ng to 1,000 mg per kg of body weight and may be
given once or more daily, weekly, monthly, or yearly. Persons of ordinary
skill in the art can readily determine optimum dosages, dosing
methodologies and repetition rates. For example, a pharmaceutical
composition or medicament comprising a small molecule compound of the
present invention is administered in a daily dose in the range from about
1 mg of small molecule compound per kg of subject weight (1 mg/kg) to
about 1 g/kg for multiple days, e.g., the daily dose is a dose in the
range of about 5 mg/kg to about 500 mg/kg, about 10 mg/kg to about 250
mg/kg, or about 25 mg/kg to about 150 mg/kg. The daily dose is
administered once per day or divided into subdoses and administered in
multiple doses, e.g., twice, three times, or four times per day.

[0141] To achieve the desired therapeutic effect, a small molecule
compound is typically administered for multiple days at the
therapeutically effective daily dose. Thus, therapeutically effective
administration of a small molecule compound to treat cancer in a subject
often requires periodic (e.g., daily) administration that continues for a
period ranging from three days to two weeks or longer. Typically, a small
molecule compound will be administered for at least three consecutive
days, often for at least five consecutive days, more often for at least
ten, and sometimes for 20, 30, 40 or more consecutive days. While
consecutive daily doses are a preferred route to achieve a
therapeutically effective dose, a therapeutically beneficial effect can
be achieved even if the small molecule compound is not administered
daily, so long as the administration is repeated frequently enough to
maintain a therapeutically effective concentration of the small molecule
compound in the subject. For example, one can administer the small
molecule compound every other day, every third day, or, if higher dose
ranges are employed and tolerated by the subject, once a week.

[0142] Optimum dosages, toxicity, and therapeutic efficacy of such small
molecule compounds may vary depending on the relative potency of
individual small molecule compounds and are determined by standard
pharmaceutical procedures in cell cultures or experimental animals, for
example, by determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50% of
the population). The dose ratio between toxic and therapeutic effects is
the therapeutic index and can be expressed as the ratio, LD50/ED50.
Compounds that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should be
taken to design a delivery system that targets such compounds to the site
of affected tissue to minimize potential damage to normal cells and,
thereby, reduce side effects. The SGK2 and PAK3 inhibitory compounds
described herein are characterized by minimal adverse side effects,
because they preferentially affect p53-deficient cells, e.g., tumor
cells, while sparing normal non-tumor cells.

[0143] The therapeutically effective dose is estimated initially from cell
culture assays. A dose can be formulated in animal models to achieve a
circulating plasma concentration range that includes the 1050 (the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such information
is then used to more accurately determine useful doses in humans. Levels
in plasma are measured, for example, by high performance liquid
chromatography (HPLC). In general, the dose equivalent of a small
molecule compound is from about 1 ng/kg to 100 mg/kg for a typical
subject.

Additional Chemical Terms and Definitions

[0144] As used herein, the term "alkyl" includes saturated aliphatic
groups, including straight-chain alkyl groups (e.g., methyl, ethyl,
propyl, butyl, pentyl, hexyl) and branched-chain alkyl groups (e.g.,
isopropyl, tert-butyl, isobutyl. In certain embodiments, a straight chain
or branched chain alkyl has six or fewer carbon atoms in its backbone
(e.g., C1-C6 for straight chain, C3-C6 for branched
chain), and in other embodiments four or fewer carbon atoms. Lower alkyl
groups include from 1-6 carbon atoms, thus the term "lower alkyl"
includes alkyl groups containing 1, 2, 3, 4, 5, or 6 carbon atoms.

[0146] The term "hydroxy" or "hydroxyl" includes groups with an --OH or
--O.sup.-.

[0147] The term "halogen" includes fluorine, bromine, chlorine, iodine,
etc. The term "perhalogenated" generally refers to a moiety wherein all
hydrogens are replaced by halogen atoms.

[0148] In the present specification, the structural formula of the
compound represents a certain isomer for convenience in some cases, but
the present invention includes all isomers such as geometrical isomer,
optical isomer based on an asymmetrical carbon, stereoisomer, tautomer
and the like which occur structurally and an isomer mixture and is not
limited to the description of the formula for convenience, and may be any
one of isomer or a mixture. Therefore, an asymmetrical carbon atom may be
present in the molecule and an optically active compound and a racemic
compound may be present in the present compound, but the present
invention is not limited to them and includes any one. In addition, a
crystal polymorphism may be present but is not limiting, but any crystal
form may be single or a crystal form mixture, or an anhydride or hydrate.
Further, so-called metabolite which is produced by degradation of the
present compound in vivo is included in the scope of the present
invention.

[0149] It will be noted that the structure of some of the compounds of the
invention include asymmetric (chiral) carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry are
included within the scope of the invention, unless indicated otherwise.
Such isomers can be obtained in substantially pure form by classical
separation techniques and by stereochemically controlled synthesis. The
compounds of this invention may exist in stereoisomeric form, therefore
can be produced as individual stereoisomers or as mixtures.

[0150] "Isomerism" means compounds that have identical molecular formulae
but that differ in the nature or the sequence of bonding of their atoms
or in the arrangement of their atoms in space. Isomers that differ in the
arrangement of their atoms in space are termed "stereoisomers".
Stereoisomers that are not mirror images of one another are termed
"diastereoisomers", and stereoisomers that are non-superimposable mirror
images are termed "enantiomers", or sometimes optical isomers. A carbon
atom bonded to four nonidentical substituents is termed a "chiral
center".

[0151] "Chiral isomer" means a compound with at least one chiral center.
It has two enantiomeric forms of opposite chirality and may exist either
as an individual enantiomer or as a mixture of enantiomers. A mixture
containing equal amounts of individual enantiomeric forms of opposite
chirality is termed a "racemic mixture". A compound that has more than
one chiral center has 2n-1 enantiomeric pairs, where n is the number
of chiral centers. Compounds with more than one chiral center may exist
as either an individual diastereomer or as a mixture of diastereomers,
termed a "diastereomeric mixture". When one chiral center is present, a
stereoisomer may be characterized by the absolute configuration (R or S)
of that chiral center. Absolute configuration refers to the arrangement
in space of the substituents attached to the chiral center. The
substituents attached to the chiral center under consideration are ranked
in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et
al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al.,
Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London),
612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964,
41, 116).

[0152] "Geometric Isomers" means the diastereomers that owe their
existence to hindered rotation about double bonds. These configurations
are differentiated in their names by the prefixes cis and trans, or Z and
E, which indicate that the groups are on the same or opposite side of the
double bond in the molecule according to the Cahn-Ingold-Prelog rules.

[0153] Further, the structures and other compounds discussed in this
application include all atropic isomers thereof. "Atropic isomers" are a
type of stereoisomer in which the atoms of two isomers are arranged
differently in space. Atropic isomers owe their existence to a restricted
rotation caused by hindrance of rotation of large groups about a central
bond. Such atropic isomers typically exist as a mixture, however as a
result of recent advances in chromatography techniques, it has been
possible to separate mixtures of two atropic isomers in select cases.

[0154] The terms "crystal polymorphs" or "polymorphs" or "crystal forms"
means crystal structures in which a compound (or salt or solvate thereof)
can crystallize in different crystal packing arrangements, all of which
have the same elemental composition. Different crystal forms usually have
different X-ray diffraction patterns, infrared spectral, melting points,
density hardness, crystal shape, optical and electrical properties,
stability and solubility. Recrystallization solvent, rate of
crystallization, storage temperature, and other factors may cause one
crystal form to dominate. Crystal polymorphs of the compounds can be
prepared by crystallization under different conditions.

[0155] Additionally, the compounds of the present invention, for example,
the salts of the compounds, can exist in either hydrated or unhydrated
(the anhydrous) form or as solvates with other solvent molecules.
Nonlimiting examples of hydrates include monohydrates, dihydrates, etc.
Nonlimiting examples of solvates include ethanol solvates, acetone
solvates, etc.

[0156] "Solvates" means solvent addition forms that contain either
stoichiometric or non stoichiometric amounts of solvent. Some compounds
have a tendency to trap a fixed molar ratio of solvent molecules in the
crystalline solid state, thus forming a solvate. If the solvent is water
the solvate formed is a hydrate, when the solvent is alcohol, the solvate
formed is an alcoholate. Hydrates are formed by the combination of one or
more molecules of water with one of the substances in which the water
retains its molecular state as H2O, such combination being able to form
one or more hydrate.

[0157] "Tautomers" refers to compounds whose structures differ markedly in
arrangement of atoms, but which exist in easy and rapid equilibrium. It
is to be understood that compounds of Formula I may be depicted as
different tautomers. It should also be understood that when compounds
have tautomeric forms, all tautomeric forms are intended to be within the
scope of the invention, and the naming of the compounds does not exclude
any tautomer form. Some compounds of the present invention can exist in a
tautomeric form which are also intended to be encompassed within the
scope of the present invention.

[0158] The compounds, salts and prodrugs of the present invention can
exist in several tautomeric forms, including the enol and imine form, and
the keto and enamine form and geometric isomers and mixtures thereof. All
such tautomeric forms are included within the scope of the present
invention. Tautomers exist as mixtures of a tautomeric set in solution.
In solid form, usually one tautomer predominates. Even though one
tautomer may be described, the present invention includes all tautomers
of the present compounds

[0159] A tautomer is one of two or more structural isomers that exist in
equilibrium and are readily converted from one isomeric form to another.
This reaction results in the formal migration of a hydrogen atom
accompanied by a switch of adjacent conjugated double bonds. In solutions
where tautomerization is possible, a chemical equilibrium of the
tautomers will be reached. The exact ratio of the tautomers depends on
several factors, including temperature, solvent, and pH. The concept of
tautomers that are interconvertable by tautomerizations is called
tautomerism.

[0160] Of the various types of tautomerism that are possible, two are
commonly observed. In keto-enol tautomerism a simultaneous shift of
electrons and a hydrogen atom occurs. Ring-chain tautomerism, is
exhibited by glucose. It arises as a result of the aldehyde group (--CHO)
in a sugar chain molecule reacting with one of the hydroxy groups (--OH)
in the same molecule to give it a cyclic (ring-shaped) form.

[0161] Tautomerizations are catalyzed by: Base: 1. deprotonation; 2.
formation of a delocalized anion (e.g. an enolate); 3. protonation at a
different position of the anion; Acid: 1. protonation; 2. formation of a
delocalized cation; 3. deprotonation at a different position adjacent to
the cation.

[0163] As used herein, the term "analog" refers to a chemical compound
that is structurally similar to another but differs slightly in
composition (as in the replacement of one atom by an atom of a different
element or in the presence of a particular functional group, or the
replacement of one functional group by another functional group). Thus,
an analog is a compound that is similar or comparable in function and
appearance, but not in structure or origin to the reference compound.

[0164] As defined herein, the term "derivative", refers to compounds that
have a common core structure, and are substituted with various groups as
described herein. For example, all of the compounds represented by
formula I are indole derivatives, and have formula I as a common core.

[0165] The term "bioisostere" refers to a compound resulting from the
exchange of an atom or of a group of atoms with another, broadly similar,
atom or group of atoms. The objective of a bioisosteric replacement is to
create a new compound with similar biological properties to the parent
compound. The bioisosteric replacement may be physicochemically or
topologically based. Examples of carboxylic acid bioisosteres include
acyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,
Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

[0166] A "pharmaceutical composition" is a formulation containing the
disclosed compounds in a form suitable for administration to a subject.

[0167] As used herein, the phrase "pharmaceutically acceptable" refers to
those compounds, materials, compositions, carriers, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for use
in contact with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk ratio.

[0168] "Pharmaceutically acceptable excipient" means an excipient that is
useful in preparing a pharmaceutical composition that is generally safe,
non-toxic and neither biologically nor otherwise undesirable, and
includes excipient that is acceptable for veterinary use as well as human
pharmaceutical use. A "pharmaceutically acceptable excipient" as used in
the specification and claims includes both one and more than one such
excipient. The compounds of the invention are capable of further forming
salts. All of these forms are also contemplated within the scope of the
claimed invention. For example, the salt can be an acid addition salt.
One example of an acid addition salt is a hydrochloride salt. Another
example is a hydrobromide salt.

[0169] "Pharmaceutically acceptable salt" of a compound means a salt that
is pharmaceutically acceptable and that possesses the desired
pharmacological activity of the parent compound.

[0172] It should be understood that all references to pharmaceutically
acceptable salts include solvent addition forms (solvates) or crystal
forms (polymorphs) as defined herein, of the same salt.

[0173] The pharmaceutically acceptable salts of the present invention can
be synthesized from a parent compound that contains a basic or acidic
moiety by conventional chemical methods. Generally, such salts can be
prepared by reacting the free acid or base forms of these compounds with
a stoichiometric amount of the appropriate base or acid in water or in an
organic solvent, or in a mixture of the two; generally, non-aqueous media
like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are
preferred. Lists of suitable salts are found in Remington's
Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). For
example, salts can include, but are not limited to, the hydrochloride and
acetate salts of the aliphatic amine-containing,
hydroxylamine-containing, and imine-containing compounds of the present
invention.

[0174] The compounds of the present invention can also be prepared as
prodrugs, for example pharmaceutically acceptable prodrugs. The terms
"pro-drug" and "prodrug" are used interchangeably herein and refer to any
compound which releases an active parent drug in vivo. Since prodrugs are
known to enhance numerous desirable qualities of pharmaceuticals (e.g.,
solubility, bioavailability, manufacturing, etc.) the compounds of the
present invention can be delivered in prodrug form. Thus, the present
invention is intended to cover prodrugs of the presently claimed
compounds, methods of delivering the same and compositions containing the
same. "Prodrugs" are intended to include any covalently bonded carriers
that release an active parent drug of the present invention in vivo when
such prodrug is administered to a subject. Prodrugs the present invention
are prepared by modifying functional groups present in the compound in
such a way that the modifications are cleaved, either in routine
manipulation or in vivo, to the parent compound. Prodrugs include
compounds of the present invention wherein a hydroxy, amino, sulfhydryl,
carboxy, or carbonyl group is bonded to any group that, may be cleaved in
vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy
or free carbonyl group, respectively.

[0176] "Protecting group" refers to a grouping of atoms that when attached
to a reactive group in a molecule masks, reduces or prevents that
reactivity. Examples of protecting groups can be found in Green and Wuts,
Protective Groups in Organic Chemistry, (Wiley, 2nd ed. 1991);
Harrison and Harrison et al., Compendium of Synthetic Organic Methods,
Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski, Protecting
Groups, (Verlag, 3rd ed. 2003).

[0177] For example, representative hydroxy protecting groups include those
where the hydroxy group is either acylated or alkylated such as benzyl,
and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers,
trialkylsilyl ethers and allyl ethers.

[0178] Stable compound" and "stable structure" are meant to indicate a
compound that is sufficiently robust to survive isolation to a useful
degree of purity from a reaction mixture, and formulation into an
efficacious therapeutic agent.

[0179] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and most
preferably less than about 4 kD. Small molecules can be, e.g., nucleic
acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or
other organic or inorganic molecules. Libraries of chemical and/or
biological mixtures, such as fungal, bacterial, or algal extracts, are
known in the art and can be screened with any of the assays of the
invention.

[0181] While the invention has been described in conjunction with the
detailed description thereof, the foregoing description is intended to
illustrate and not limit the scope of the invention, which is defined by
the scope of the appended claims. Other aspects, advantages, and
modifications are within the scope of the following claims.

[0182] The patent and scientific literature referred to herein establishes
the knowledge that is available to those with skill in the art. All
United States patents and published or unpublished United States patent
applications cited herein are incorporated by reference. All published
foreign patents and patent applications cited herein are hereby
incorporated by reference. Genbank and NCBI submissions indicated by
accession number cited herein are hereby incorporated by reference. All
other published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.

[0183] While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details may be
made therein without departing from the scope of the invention
encompassed by the appended claims.